Abstract
The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4–104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3–100%. Additionally, the recombination efficiencies were 2.5–45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome.
Highlights
Chromosomal modifications are critical tools for genomic and metabolic engineering [1,2,3,4]
The helper plasmid pCNA and the landing pad plasmid pBDC-Xd were transformed into E. coli DH1 cells
With the help of the λ-Red system and I-CreI, the homologous fragment generated by double-strand breaks (DSBs) was integrated into the genome to replace the nonessential region (Fig 3a-1 and 3b-1)
Summary
Chromosomal modifications are critical tools for genomic and metabolic engineering [1,2,3,4]. In Escherichia coli, the λ-Red recombination system can be used for chromosomal modifications, including gene deletions, mutations, and integration. This system is constituted by three proteins: Exo, Gam, and Beta. Exo protein binds to the end of linear DNA and generates 30 overhangs, whereas Gam binds to the RecBCD complex, thereby preventing the degradation of double-stranded DNA. Beta mediates annealing between complementary strands [5,6,7].
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